1. Field of the Invention
The invention relates to a device consisting of an optical waveguide, which at least has a diffractive element for coupling excitation radiation into the wave-guiding layer and on the wave-guiding layer of which there is located a second, tightly sealing layer made from a material which, at least at the support surface in the region of the guided excitation radiation, is transparent both to the excitation radiation and to the evanescently excited radiation at least to the penetration depth of the evanescent field, and which has, at least in a partial region of the guided excitation radiation, a cavity for an analysis sample, the depth of the cavity corresponding at least to the penetration depth of the evanescent field, and wherein the diffractive element is fully covered by the material of the second layer in the coupling-in region of the excitation radiation. The invention relates also to a method that uses the device for detecting, in an analyte sample, molecules that are capable of being evanescently excited to luminescence.
2. Description of Related Art
Planar waveguides for generating and detecting evanescently excited radiation have recently been developed especially in the area of biochemical analytical science. In the evanescent field, on contact with an analyte sample, luminescence, for example fluorescence, is generated, the measurement of which allows the qualitative and quantitative determination of substances even when they are present in very low concentrations. The evanescently excited radiation emitted isotropically into space is determined by means of suitable measuring devices, such as photodiodes, photomultipliers or CCD cameras. That method is disclosed, for example, in WO 95/33197. It is also possible for the portion of evanescently excited radiation coupled back into the waveguide to be coupled out by means of a diffractive optical element, for example a grating, and measured. That method is described, for example, in WO 95/33198.
In affinity sensory analysis, for specific identification of an analyte in a sample, which may consist of a complex mixture of substances, and for binding the analyte molecules to the surface of the waveguide, in the region of the penetration depth of the evanescent field, biochemical recognition elements are immobilized on the waveguide surface either directly or by means of an adhesion-imparting layer. For the purpose of detecting the analyte, the dissolved sample is brought into contact, either in intermittent flow or continuous flow, with the recognition elements immobilized on the waveguide surface.
A problem that arises when using measuring cells in which the sample liquid comes into contact with the diffractive coupling-in elements is that the conditions for coupling-in of the excitation light may change as a result of molecular adsorption or binding onto the coupling-in elements. Furthermore, since unbound luminescent or fluorescent molecules are excited by the portion of excitation light that is not coupled into the waveguide but enters the solution unrefracted at zero order, background luminescence or fluorescence may be excited deep within the sample, some of which may be coupled into the waveguide via the coupling-in grating and may impair the accuracy and sensitivity of the determination of the analyte.
Isolating the coupling-in element from the region of contact with the sample by means of materials on which no special demands are placed in terms of transparency and refractive index may result in significant impairment in guidance of the light, possibly even in its entire suppression, in the region of contact with the sample, which is the region relevant to the measurement. That problem is described in greater detail in WO 97/01087.
In order to reduce those disadvantages, WO 97/01087 describes a counter-flow cell in which a transparent reference liquid, which is guided in countercurrent to the analyte sample and does not interact specifically with the recognition elements, is used to produce a sample-free blocking volume in the region of coupling-in elements of optical waveguides, so that constant conditions are obtained in the coupling-in region of the excitation radiation. However, that arrangement, improved especially for the measurement of evanescently excited radiation, is technically relatively complex, is scarcely feasible for intermittent flow applications and accordingly is poorly suited, from the point of view of ease of operation, to routinely use in, for example, diagnostic devices.
Analytical Chemistry, Volume 62, No. 18(1990), pages 2012-2017, describes a flow-through flow cell made from silicone rubber and applied to an optical waveguide having a coupling-in grating and a coupling-out grating. The coupling-in element and coupling-out element are located in the region of the sample flow channel. Using that arrangement, changes in light absorption and refractive index are measured without selective interaction with specific recognition elements at the waveguide surface. In the case of the analyte (a dye solution in the case of absorption-dependent measurement, or liquids having different refractive indices in the case of refractive index-dependent measurement) adsorption phenomena on the surface are disregarded. In those very insensitive measurements, the changes in the effective refractive index that are to be expected for a mode guided in the waveguide are in fact negligible compared with the large changes in the refractive indices of the solutions fed inxe2x80x94even in the event of a monolayer of molecules being adsorbedxe2x80x94which is contrary to the disruptions to be expected in the very much more sensitive method of determining the luminescence generated in the evanescent field. Of course, in the case of the refractive index-dependent measuring method based on the change in the coupling-in or coupling-out angle, contact between the sample and the coupling elements is actually necessary in order to generate the measurement signal. As a result of that configuration, which has a coupling-in element and coupling-out element located within the sample flow channel, the sample cell merely has the task of providing a seal against the efflux of liquid without any further demands being placed on optical properties of the material.
In order to carry out analyte determinations that are based on measuring the luminescence generated in the evanescent field of a waveguide, a device is therefore required by means of which radiation evanescently excited through a planar optical waveguide can be determined with a high degree of consistency and measurement accuracy, the measuring device at the same time being easy to produce and easy to operate.
It has now been found, surprisingly, that
a) a high degree of measurement accuracy is achieved,
b) an excellent degree of measurement consistency is achieved,
c) a high degree of measurement sensitivity is achieved,
d) it is possible for the device filled with analyte molecules, especially immobilised analyte molecules, to be stored for a relatively long time, and
e) devices that are easy to operate in terms of measurement technique are made available, when the device comprising a waveguide and sample receptacle is so configured that the diffractive element for coupling in excitation radiation is fully covered, at least in the coupling-in region of the excitation radiation, by a layer that is transparent to the excitation radiation and to the evanescently excited radiation.
In view of the fact that the recess is arranged downstream of the coupling-in element in the direction of propagation of excitation radiation and that the diffractive element (coupling-in element) is covered by a layer forming the recess, constant coupling-in conditions are obtained for the excitation radiation in the coupling-in element. On the other hand, abrupt changes in refractive index in the region of penetration of excitation radiation into the material adjacent to the waveguide are minimized to a very large extent. Substantially disruption-free guidance of the excitation radiation and evanescently excited and coupled-back radiation in the waveguide is achieved, which results in a high signal yield and, for example, even under investigation conditions that are frequently not entirely optimal in routine use yields analytical results that can still be utilized. Disruptive abrupt changes in refractive index are also suppressed by means of the rounding.
The invention relates firstly to a device comprising a planar optical waveguide, including a transparent carrier (40) and a wave-guiding layer (41), wherein the waveguide at least has a diffractive element (42) for coupling excitation radiation into the wave-guiding layer. Located on the wave-guiding layer is a further, tightly sealing layer (43) made from a material which, at least at the support surface, is transparent both to the excitation radiation and to the evanescently excited radiation at least to the penetration depth of the evanescent field, and which, for an analysis sample, has, at least in a partial region of the guided excitation radiation, a cavity (45) which is open to the upper side or a cavity (6) which is closed to the upper side and connected by means of an inflow channel (2) and an outflow channel (3). The depth of the cavity corresponds at least to the penetration depth of the evanescent field, and the diffractive element (42) is fully covered by the material of the layer (43) at least in the coupling-in region of the excitation radiation.
Optical waveguides having one or two diffractive elements for coupling in the excitation radiation and coupling out the luminescence radiation and the configuration thereof for the determination of analyte molecules according to the fluorescence method are known and are described, for example, in WO 95/33197 and WO 95/33198. Diffractive elements are to be understood as being coupling-in and coupling-out elements for light radiation. Gratings, which may be produced in various ways, are frequently used. For example, such gratings may be arranged in the transparent carrier or the wave-guiding layer and impressed during their formation or subsequently. It is also possible to produce such gratings by means of ablative techniques (laser irradiation). Other production techniques are holographic recordings or implantation of ions by means of ion bombardment.
The layer (43) forming a cavity is, at least at the support surface, transparent to electro-magnetic radiation in the range of the excitation wavelength and the luminescence wave-length. It may be made from an inorganic material, such as, for example, glass or quartz, or transparent organic polymers (organic glasses), such as, for example, polyesters, polycarbonates, polyacrylates, polymethacrylates or photopolymerisates. The layer (43) is preferably formed from an elastomer. Elastomers of polysiloxanes, such as polydimethylsiloxanes, which are soft and pliable and are frequently self-adhering materials, are especially suitable. The materials for the layer (43) are known and, in some instances, commercially available.
The layer (43) having at least one cavity can be produced by means of conventional forming techniques, for example casting and pressing techniques, or by means of grinding, stamping and milling techniques starting from appropriately pre-formed semi-finished products. The layer (43) may alternatively comprise photopolymerisable substances, which can be applied directly to the wave-guiding layer by means of photolithographic techniques.
Where surfaces are very smooth (surface roughness in the nanometer range or below), in the case of rigid materials self-adhesion may, on adhesion, result in tight seals. Elastomers are, in general, self-adhesive. Surface roughness that is as low as possible is also very desirable in order to suppress any scattering of light. In those cases, the layer (43) is preferably produced as a separate body and applied, in tightly sealing contact, to the wave guide, on the surface of which there may be located immobilized recognition elements, where appropriate on an additional thin (i.e.  less than 100 nm) adhesion-imparting layer.
The layer (43) may comprise a single material that is transparent and luminescence-free at least at the excitation wavelength and luminescence wavelength of the analyte or may alternatively be in the form of a layer having two strata, of which the first, which is brought into contact with the waveguide surface, must be transparent and luminescence-free at the excitation wavelength and luminescence wavelength of the analyte, while the covering layer, adjacent thereto, is preferably radiation-absorbing. In that arrangement, the thickness of the first layer in contact with the waveguide surface encompasses at least the penetration depth (44) of the evanescent field, i.e. at least approximately 0.5 xcexcm. The thickness of that first layer is preferably from 0.5 xcexcm to 10 mm, especially from 0.01 mm to 10 mm.
The depth of the cavities may correspond at least to the penetration depth of the evanescent field, that is to say approximately 0.5 xcexcm. In general, the depth is preferably from 0.5 xcexcm to 10 mm, especially from 0.01 to 10 mm, more especially from 0.05 to 5 mm and very especially from 0.05 to 2 mm.
In an advantageous arrangement, abrupt changes in refractive index between the wave-guiding layer (41) and the layer 43 are avoided, this being achieved by providing the boundary of the cavity with a rounded configuration at the support perpendicular to the wave-guiding layer. A rounded transition perpendicular to the surface of the waveguide, at the boundaries of the cavities, means that a right angle is avoided. The rounding may be, for example, part of a circular, parabolic or hyperbolic curve. When soft and pliable materials are used for the layer (43), the rounding is formed automatically by the act of pressing the layer onto the waveguide. The rounding may, however, alternatively be pre-formed by the forming process. Abrupt changes in refractive index can also be avoided by the cavity being made narrower, where appropriate continuously, in the direction of propagation of the excitation light. Another possibility consists in selecting for the layer (43) a material having a refractive index close to or equal to the refractive index of the analyte sample.
The evanescently excited radiation emitted isotropically into space can be determined by means of suitable measuring devices. The device according to the invention may, however, also comprise a second diffractive element in the waveguide for coupling out radiation that has been evanescently excited and coupled back into the waveguide. Advantageously, that diffractive element, too, is fully covered, at least in the coupling-out region, by the material of the layer (43) and advantageously the transition of the layer (43) perpendicular to the waveguide surface is, in that case too, rounded at least towards that diffractive element.
In a further development of the device according to the invention multiple cavities are provided so that more than one, for example from 2 to 100 or more, preferably from 4 to 100 cavities are provided, which can be arranged in the longitudinal or transverse direction relative to the propagation of the excitation radiation. For example, cavities may be arranged, in one row after another, in a transverse direction relative to the propagation of the excitation radiation, extending from a diffractive coupling-in element to a point upstream of a diffractive coupling-out element, which may be present; in that case, there are provided preferably from 2 to 10, and especially from 2 to 5, cavities. Another possibility includes providing a plurality of individual diffractive elements in a row or one long diffractive element and arranging the cavities parallel to (that is to say in the longitudinal direction relative to) the direction of propagation of the excitation light. In that case, 100 or more cavities can be present. Furthermore, it is possible for 2 or more, for example from 2 to 50, especially from 2 to 20, of the multiple arrangements to be provided on the surface of a large planar waveguide. Those multiplication possibilities allow flexible adaptation of the device according to the invention to practical requirements. Such further developments are suitable especially for series, comparative and parallel measurements, and also for automated measurement arrangements, using suitably configured measuring heads.
Where there are multiple arrangements in the longitudinal direction relative to (parallel to) the propagation of the excitation radiation it may be advantageous to provide light-absorbing materials, for example dyes, pigments, soot, metal oxides or metals, along the cavities for the purpose of suppressing scattered radiation. Such materials may be located in additional cavities provided for that purpose in the longitudinal direction relative to the direction of propagation of the excitation light or may be applied in the form of a sheet to the surface of the wave-guiding layer in the longitudinal direction relative to the direction of propagation of the excitation light. Arrangements in the form of sheets, which are simple to produce by means of coating or vapor-deposition techniques, are advantageous. The absorbing materials may also be applied above the diffractive elements, leaving the coupling-in region free. The device may, for example, be so configured that attenuating material that absorbs in the spectral range of excitation radiation (18) and of evanescently excited radiation (21) is provided between the flow cell (1, 26, 30, 35, 38) and the waveguide (7) on both sides of the or each recess (6), or that the attenuating material (27) is applied in the form of a sheet as an immersion, or that attenuating recesses (33), which can be filled with attenuating material, are provided. Devices having cavities open to the upper side can be configured in the same manner.
The device according to the invention may be provided in various embodiments, a distinction being drawn between embodiments having an open cavity (embodiment A) and those having a closed cavity (embodiment B, flow-through cells).
Embodiment A
The open recesses may be of any shape desired per se; they may be, for example, square, rectangular, round or ellipsoidal. The configuration of devices according to the invention can, for example, correspond to the shape of known microtitre plates. The geometric arrangement of the recesses is any that is desired per se, preference being given to arrangements in rows. Devices and preferences illustrated and described for embodiment B can also be applied analogously to embodiment A.
Embodiment B
In a device according to the invention for generating evanescently excited radiation, which device has at least one coupling-out element-for coupling evanescently excited radiation out of the waveguide, it is advantageous for the flow cell also to cover the one or each coupling-out element. In a further development in that connection, provision is made for the recess to be arranged in its entirety between the one or each coupling-in element and the one or each coupling-out element so that both the one or each coupling-in element and the one or each coupling-out element are free of sample material. This has the advantage that constant coupling conditions, uninfluenced by sample material, prevail during both the coupling-in and the coupling-out of excitation radiation and of evanescently excited radiation.
In further developments, a plurality of recesses are provided which are arranged in the longitudinal or transverse direction relative to the direction of propagation of excitation radiation. When recesses are aligned parallel to the direction of propagation of excitation radiation it is advantageous to provide, between the recesses in the penetration region of excitation radiation and evanescently excited radiation, material that absorbs in the spectral range of those radiations, for example from the ultraviolet to the infrared spectral range, in order to prevent portions of radiation being cross-coupled between the recesses. That may be accomplished, for example, by means of an absorbing layer applied between the flow cell and the waveguide. In another example embodiment, there are incorporated in the flow cell, between a plurality of recesses aligned parallel to the direction of propagation of the excitation radiation, attenuating recesses, which can be filled with a radiation-absorbing liquid and which are open towards the same surface side as the recesses.
For use in routine analytical operation it is also advantageous for the flow cell to comprise a pliable material that tightly seals the at least one recess when applied to the waveguide. In that way it is possible without further aids, such as seals, by mounting a flow cell on the waveguide, for sample material to be passed through the flow cell without leaks.